Coleopteran-active Bacillus thuringiensis toxin

ABSTRACT

The subject invention concerns a novel microbe and gene encoding a novel toxin protein with activity against insect pests of the order Coleoptera. Pests in the order Coleoptera do heavy damage to crops, e.g., corn. The novel Bacillus thuringiensis microbe of the invention is referred to as B.t. PS50C. The spores or crystals of this microbe, or mutants thereof, are useful to control coleopteran pests in various environments. The novel gene of the invention can be used to transform various hosts wherein the novel toxic protein can be expressed.

This is a division of application Ser. No. 07/642,112, filed Jan. 16,1991, now U.S. Pat. No. 5,277,905.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (B.t.) produces an insect toxin designated asδ-endotoxin. It is synthesized by the B.t. sporulating cell. The toxin,upon being ingested in its crystalline form by susceptible insectlarvae, is transformed into biologically active moieties by the insectgut juice proteases. The primary target is insect cells of the gutepithelium, which are rapidly destroyed.

The reported activity spectrum of B.t. covers insect species within theorder Lepidoptera, many of which are major pests in agriculture andforestry. The activity spectrum also includes the insect order Diptera,which includes mosquitos and black flies. See Couch, T. L. (1980)"Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis,"Developments in Industrial Microbiology 22:61-76; Beegle, C. C., (1978)"Use of Entomogenous Bacteria in Agroecosystems," Developments inIndustrial Microbiology 20:97-104. Krieg, et al., Z. ang. Ent. (1983)96:500-508, describe a B.t. isolate named Bacillus thuringiensis var.tenebrionis, which is reportedly active against two beetles in the orderColeoptera. These are the Colorado potato beetle, Leptinotarsadecemlineata, and Agelastica alni.

In European Patent Application 0 202 739 there is disclosed a novel B.t.isolate active against Coleoptera. It is known as B. thuringiensis var.san diego (B.t.sd.). U.S. Pat. No. 4,966,765, discloses thecoleopteran-active Bacillus thuringiensis isolate B.t. PS86B1. EuropeanPatent Application 0 337 604 also discloses a novel B.t. isolate activeagainst Coleoptera. This isolate is B.t. PS43F.

Coleopteran-active strains, such as B.t.sd., B.t. PS86B1, and B.t.PS43F, can be used to control foliar-feeding beetles. The Coloradopotato beetle (Leptinotarsa decemlineata), for example, is susceptibleto the delta-endotoxin of B.t.sd. and larvae are killed upon ingesting asufficient dose of spore/crystal preparation on treated foliage.

A number of crops are attacked by flea beetles. These beetles belong tothe family Chrysomelidae, the decemlineata. The adults can causeextensive damage by feeding on the foliage.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a novel Bacillus thuringiensis (B.t.)isolate and a cloned gene therefrom which encodes a novelcoleopteran-active protein. The novel B.t. isolate, known herein asBacillus thuringiensis PS50C (B.t. PS50C), has thus far been shown to beactive against the Colorado potato beetle (Leptinotarsa decemlineata).The novel δ-endotoxin gene of the invention encodes an ≈130 kDa protein.

The subject invention also includes mutants of B.t. PS50C which havesubstantially the same pesticidal properties as B.t. PS50C. Proceduresfor making mutants are well known in the microbiological art.Ultraviolet light and nitrosoguanidine are used extensively toward thisend.

Further, the invention also includes the treatment of substantiallyintact B.t. PS50C cells, and recombinant cells containing the gene ofthe invention, to prolong the pesticidal activity when the substantiallyintact cells are applied to the environment of a target pest. Suchtreatment can be by chemical or physical means, or a combination ofchemical or physical means, so long as the technique does notdeleteriously affect the properties of the pesticide, nor diminish thecellular capability in protecting the pesticide. The treated cell actsas a protective coating for the pesticidal toxin. The toxin becomesavailable to act as such upon ingestion by a target insect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--Photograph of a Standard SDS Polyacrylamide Gel of B.t. PS50C,B.t.sd., and B.t. PS86B1.

FIG. 2--Restriction map of pMYC1638.

DETAILED DISCLOSURE OF THE INVENTION

The novel Bacillus thuringiensis isolate of the subject invention hasthe following characteristics in its biologically pure form:

Characteristics of B.t. PS50C

Colony morphology--Large colony, dull surface, typical B.t.

Vegetative cell morphology--typical B.t.

Culture methods--typical for B.t.

Flagellar serotyping--PS50C belongs to serotype 18, kumamotoensis.

Crystal morphology--a sphere.

RFLP analysis--Southern hybridization of total DNA distinguishes B.t.PS50C from B.t.sd. and other B.t. isolates.

Alkali-soluble proteins--SDS polyacrylamide gel electrophoresis(SDS-PAGE) shows a 130 kDa doublet protein.

A comparison of the characteristics of B. thuringiensis PS50C (B.t.PS50C) to the characteristics of the known B.t. strains B. thuringiensisvar. san diego (B.t.sd.), B. thuringiensis PS86B1 (NRRL B-18299), and B.thuringiensis var. kurstaki (HD-1) is shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Comparison of B.t. PS50C, B.t. PS86B1, B.t.sd., and B.t. HD-1                          B.t. PS50C                                                                            B.t.sd.                                                                             B.t. PS86B1                                                                          B.t. HD-1                                       __________________________________________________________________________    Serovar  kumamotoensis                                                                         morrisoni                                                                           tolworthi                                                                            kurstaki                                        Type of inclusion                                                                      sphere  square                                                                              flat, pointed                                                                        Bipyramid                                                        wafer ellipse, plus                                                                 sm. inclusions                                         Size of alkali-                                                                        130 kDa 72,000                                                                              75,000 130,000                                         soluble proteins                                                                       doublet 64,000                                                                              68,000 68,000                                          by SDS-PAGE            61,000                                                 Host range                                                                             Coleoptera                                                                            Coleoptera                                                                          Coleoptera                                                                           Lepidoptera                                     __________________________________________________________________________

The cultures disclosed in this application have been deposited in theAgricultural Research Service Patent Culture Collection (NRRL), NorthernRegional Research Center, 1815 North University Street, Peoria, Ill.61604, USA.______________________________________Culture Repository No.Deposit date______________________________________Bacillus thuringiensisNRRL B-18746 January 9, 1991PS50CEscherichia coli NM522 NRRL B-18751January 11, 1991[pMYC1638]______________________________________

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C.122. The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of a deposit, and in any case, for a period of at least thirty(30) years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposits. All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

B.t. PS50C, NRRL B-18746, can be cultured using standard art media andfermentation techniques. Upon completion of the fermentation cycle, thebacteria can be harvested by first separating the B.t. spores andcrystals from the fermentation broth by means well known in the art. Therecovered B.t. spores and crystals can be formulated into a wettablepowder, liquid concentrate, granules, or other formulations by theaddition of surfactants, dispersants, inert carriers and othercomponents to facilitate handling and application for particular targetpests. These formulation and application procedures are all well knownin the art.

Plasmid DNA (pMYC1638) containing the toxin gene from B.t. PS50C can bepurified from E. coli NM522[pMYC1638] by standard procedures well knownin the art. The toxin gene can be excised from the plasmid DNA byrestriction enzyme digestion, as indicated in FIG. 2.

Formulated products can be sprayed or applied onto foliage to controlphytophagous beetles or caterpillars.

Another approach that can be taken is to incorporate the spores andcrystals of B.t. PS50C into bait granules containing an attractant andapplying these granules to the soil for control of soil-inhabitingColeoptera. Formulated B.t. PS50C can also be applied as a seed-coatingor root treatment or total plant treatment.

The B.t. PS50C cells can be treated prior to formulation to prolong thepesticidal activity when the cells are applied to the environment of atarget pest. Such treatment can be by chemical or physical means, or bya combination of chemical and/or physical means, so long as thetechnique does not deleteriously affect the properties of the pesticide,nor diminish the cellular capability in protecting the pesticide.Examples of chemical reagents are halogenating agents, particularlyhalogens of atomic no. 17-80. More particularly, iodine can be usedunder mild conditions and for sufficient time to achieve the desiredresults. Other suitable techniques include treatment with aldehydes,such as formaldehyde and glutaraldehyde; anti-infectives, such aszephiran chloride; alcohols, such as isopropyl and ethanol; varioushistologic fixatives, such as Bouin's fixative and Helly's fixative(See: Humason, Gretchen. L., Animal Tissue Techniques, W. H. Freeman andCompany, 1967); or a combination of physical (heat) and chemical agentsthat prolong the activity of the toxin produced in the cell when thecell is applied to the environment of the target pest(s). Examples ofphysical means are short wavelength radiation such as gamma-radiationand X-radiation, freezing, UV irradiation, lyophilization, and the like.

The novel toxin gene of the subject invention was obtained from a novelcoleopteran-active B. thuringiensis (B.t.) isolate designated B.t.PS50C. The gene was isolated as disclosed in the Examples.

The toxin gene of the subject invention can be introduced into a widevariety of microbial hosts. Expression of the toxin gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the situs of coleopteran insects where they willproliferate and be ingested by the insects. The result is a control ofthe unwanted insects. Alternatively, the microbe hosting the toxin genecan be treated under conditions that prolong the activity of the toxinproduced in the cell. The treated cell then can be applied to theenvironment of target pest(s). The resulting product retains thetoxicity of the B.t. toxin.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the "phytosphere"(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus., Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffiuens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

A wide variety of ways are available for introducing the B.t. geneexpressing the toxin into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. One can providefor DNA constructs which include the transcriptional and translationalregulatory signals for expression of the toxin gene, the toxin geneunder their regulatory control and a DNA sequence homologous with asequence in the host organism, whereby integration will occur, and/or areplication system which is functional in the host, whereby integrationor stable maintenance will occur.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In some instances, it may bedesirable to provide for regulative expression of the toxin, whereexpression of the toxin will only occur after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger, particularly by using an active promoter, as well as byemploying sequences, which enhance the stability of the messenger RNA.The initiation and translational termination region will involve stopcodon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5' or 3' of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon(s), thepolyadenylation signal sequence, if any, and the terminator region. Thissequence as a double strand may be used by itself for transformation ofa microorganism host, but will usually be included with a DNA sequenceinvolving a marker, where the second DNA sequence may be joined to thetoxin expression construct during introduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototropy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the toxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the toxin-producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, and usually not more than about 1000 bp of asequence homologous with a sequence in the host. In this way, theprobability of legitimate recombination is enhanced, so that the genewill be integrated into the host and stably maintained by the host.Desirably, the toxin gene will be in close proximity to the geneproviding for complementation as well as the gene providing for thecompetitive advantage. Therefore, in the event that a toxin gene islost, the resulting organism will be likely to also lose thecomplementing gene and/or the gene providing for the competitiveadvantage, so that it will be unable to compete in the environment withthe gene retaining the intact construct.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the lambda left and right promoters, the tac promoter,the naturally-occurring promoters associated with the toxin gene, wherefunctional in the host. See for example, U.S. Pat. Nos. 4,332,898,4,342,832 and 4,356,270. The termination region may be the terminationregion normally associated with the transcriptional initiation region ora different transcriptional initiation region, so long as the tworegions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pRO1614, and the like. See for example, Olson et al., (1982) J.Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, andU.S. Pat. Nos. 4,356,270, 4,362,817, and 4,371,625.

The B.t. gene can be introduced between the transcriptional andtranslational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants then can be tested forpesticidal activity.

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thentreated cell is applied to the environment of target pest(s), mayinclude either prokaryotes or eukaryotes, normally being limited tothose cells which do not produce substances toxic to higher organisms,such as mammals. However, organisms which produce substances toxic tohigher organisms could be used, where the toxin is unstable or the levelof application sufficiently low as to avoid any possibility of toxicityto a mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and- positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion; ease of killing and fixingwithout damage to the toxin; and the like. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., Streptomyces sp., and the like.Specific organisms include Pseudomonas aeruginosa, Pseudomonasfluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis,Escherichia coli, Bacillus subtilis, Streptomyces lividans, and thelike.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the recombinant microbial cell can be done as disclosedinfra. The treated cells generally will have enhanced structuralstability which will enhance resistance to environmental conditions.Where the pesticide is in a proform, the method of inactivation shouldbe selected so as not to inhibit processing of the proform to the matureform of the pesticide by the target pest pathogen. For example,formaldehyde will crosslink proteins and could inhibit processing of theproform of a polypeptide pesticide. The method of inactivation orkilling retains at least a substantial portion of the bio-availabilityor bioactivity of the toxin.

The cellular host containing the B.t. insecticidal gene may be grown inany convenient nutrient medium, where the DNA construct provides aselective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells may be formulated in a variety of ways. They may beemployed as wettable powders, granules or dusts, by mixing with variousinert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the natureof the particular formulation, particularly whether it is a concentrateor to be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the coleopteranpest(s), e.g., plants, soil or water, by spraying, dusting, sprinkling,or the like.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1--Culturing B.t. PS50C, NRRL B-18746

A subculture of B.t. PS50C, NRRL B-18746 can be used to inoculate thefollowing medium, a peptone, glucose, salts medium.

    ______________________________________                                        Bacto Peptone          7.5    g/l                                             Glucose                1.0    g/l                                             KH.sub.2 PO.sub.4      3.4    g/l                                             K.sub.2 HPO.sub.4      4.35   g/l                                             Salt Solution          5.0    ml/l                                            CaCl.sub.2 Solution    5.0    ml/l                                            Salts Solution (100 ml)                                                       MgSO.sub.4.7H.sub.2 O  2.46   g                                               MnSO.sub.4.H.sub.2 O   0.04   g                                               ZnSO.sub.4.7H.sub.2 O  0.28   g                                               FeSO.sub.4.7H.sub.2 O  0.40   g                                               CaCl.sub.2 Solution (100 ml)                                                  CaCl.sub.2.2H.sub.2 O  3.66   g                                               pH 7.2                                                                        ______________________________________                                    

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and crystals, obtained in the above fermentation, can beisolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2--Testing of B.t. PS50C, NRRL B-18746 Spores and Crystals

B.t. PS50C, NRRL B-18746 spores and crystals are toxic to the Coloradopotato beetle (CPB). The assay for the Colorado potato beetle wasconducted as follows:

CPB Bioassay--Early second instar larvae of Leptinotarsa decemlineataare placed on potato leaves which have been dipped in suspensionscontaining Bacillus thuringiensis preparations. The larvae are incubatedat 25° C. for 4 days, and larval mortality is recorded and analyzedusing probit analysis.

EXAMPLE 3--Cloning of a Novel Toxin Gene from B.t. Isolate PS50C

Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cellsgrown to an optical density, at 600 nm, of 1.0. The cells were recoveredby centrifugation and protoplasts were prepared in TES buffer (30 mMTris-HCl, 10 mM EDTA, 50 mM NaCl, pH=8.0) containing 20% sucrose and 50mg/ml lysozyme. The protoplasts were lysed by addition of SDS to a finalconcentration of 4%. The cellular material was precipitated overnight at4° C. in 100 mM (final concentration) neutral potassium chloride. Thesupernate was extracted twice with phenol/chloroform (1:1). Nucleicacids were precipitated with ethanol and DNA was purified by isopycnicbanding on cesium chloride-ethidium bromide gradients.

Total cellular DNA from B.t. subsp. kumamotoensis (B.t.kum.), isolatePS50C, was digested with HindIll and fractionated by electrophoresis ona 0.8% (w/v) agarose-TAE (50 mM Tris-HCl, 20 mM NaOAc, 2.5 mM EDTA,pH=8.0) buffered gel. A Southern blot of the gel was hybridized with a[32P]-radiolabeled oligonucleotide probe. Results showed that thehybridizing fragments of PS50C are approximately 12 Kb and 1.7 Kb insize.

A library was constructed from PS50C total cellular DNA partiallydigested with Sau3A and size fractionated by gel electrophoresis. The9-23 Kb region of the gel was excised and the DNA was electroeluted andthen concentrated using an Elutip-d™ ion exchange column (Schleicher andSchuel, Keene, N.H.). The isolated Sau3A fragments were ligated intoBamHI-digested LambdaGEM-11™ (PROMEGA). The packaged phage were platedon E. coli KW251 cells (PROMEGA) at a high titer and screened using theradiolabeled oligonucleotide probe. Hybridizing plaques were purifiedand rescreened at a lower plaque density. Single isolated, purifiedplaques that hybridized with the probe were used to infect E. coli KW251 cells in liquid culture for preparation of phage for DNA isolation.DNA was isolated by standard procedures. Preparative amounts of DNA weredigested with XhoI (to release the inserted DNA from lambda sequences)and separated by electrophoresis on a 0.6% agarose-TAE gel. The largefragments were purified by ion exchange chromatography as above andligated to XhoI-digested, dephosphorylated pHTBlueII (an E. coli/B.thuringiensis shuttle vector comprised of pBluescript s/k [Stratagene]and the replication origin from a resident B.t. plasmid [D. Lereclus etal. 1989. FEMS Microbiology Letters 60:211-218]). The ligation mix wasintroduced by transformation into competent E. coli NM522 cells (ATCC47000) and plated on LB agar containing ampicillin,isopropyl-(β)-D-thiogalactoside (IPTG) and5-bromo-4-chloro-4-indolyl-(β)-D-galactoside (XGAL). White colonies,with putative restriction fragment insertions in the (β)-galactosidasegene of pHTBlueII, were subjected to standard rapid plasmid purificationprocedures. Plasmids were analyzed by XhoI digestion and agarose gelelectrophoresis. The desired plasmid construct, pMYC1638, contains anapproximately 12 Kb XhoI insert. A partial restriction map (FIG. 2) ofthe cloned insert indicates that the toxin gene is novel compared to themaps of other toxin genes encoding insecticidal proteins.

Plasmid pMYC1638 was introduced into an acrystalliferous (Cry⁻) B.t.host (HD-1 cryB obtained from A. Aronson, Purdue University) byelectroporation. Expression of an approximately 130 kDa protein wasverified by SDS-PAGE. Broth containing spores and crystals was used forthe determination of toxicity to Leptinotarsa decemlineata.

Plasmid pMYC1638 containing the B.t. toxin gene, can be removed from thetransformed host microbe by use of standard well-known procedures. Forexample, E. coli NM522[pMYC1638] NRRL B-18751 can be subjected tocleared lysate isopycnic density gradient procedures, and the like, torecover pMYC1638.

EXAMPLE 4--Insertion of Toxin Gene Into Plants

The novel gene coding for the novel insecticidal toxin, as disclosedherein, can be inserted into plant cells using the Ti plasmid fromAgrobacter tumefaciens. Plant cells can then be caused to regenerateinto plants (Zambryski, P., Joos, H., Gentello, C., Leeroans, J., VanMontague, M. and Schell, J [1983] Cell 32:1033-1043). A particularlyuseful vector in this regard is pEND4K (Klee, H. J., Yanofsky, M. F. andNester, E. W. [1985] Bio/Technology 3:637-642). This plasmid canreplicate both in plant cells and in bacteria and has multiple cloningsites for passenger genes. The toxin gene, for example, can be insertedinto the BamHI site of pEND4K, propagated in E. coli, and transformedinto appropriate plant cells.

EXAMPLE 5--Cloning of Novel B. thuringiensis Gene Into Baculoviruses

The novel gene of the invention can be cloned into baculoviruses such asAutographa californica nuclear polyhedrosis virus (AcNPV). Plasmids canbe constructed that contain the AcNPV genome cloned into a commercialcloning vector such as pUC8. The AcNPV genome is modified so that thecoding region of the polyhedrin gene is removed and a unique cloningsite for a passenger gene is placed directly behind the polyhedrinpromoter. Examples of such vectors are pGP-B6874, described by Pennocket al. (Pennock, G. D., Shoemaker, C. and Miller, L. K. [1984] Mol.Cell. Biol. 4:399-406), and pAC380, described by Smith et al. (Smith, G.E., Summers, M. D. and Fraser, M. J. [1983] Mol Cell. Biol.3:2156-2165). The gene coding for the novel protein toxin of theinvention can be modified with BamHI linkers at appropriate regions bothupstream and downstream from the coding region and inserted into thepassenger site of one of the AcNPV vectors.

We claim:
 1. A toxin active against coleopteran pests, isolated andpurified from Bacillus thuringiensis PS50C, having the identifyingcharacteristics of NRKL B-18746, wherein said toxin has a molecularweight of ≈130 kDa.